Horseshoe crabs have a type of hemocyte that is filled with two kinds of granules, large and small, having different densities. The large granules contain a body fluid coagulation factor and an anti-lipopolysaccharide factor, and the small granules contain antimicrobial substances such as tachyplesin. The inventors of the present invention isolated six proteins, designated S1-S6, from the hemocytes of horseshoe crab which are components of small granules (Shigenaga, T. J. et al., J. Biochem., 114, 307-316(1993)).
A group of peptides having antimicrobial activity and generally called defensins have been previously isolated from cellular granitles of neutrophils and macrophages of mammals such as humans, rabbits, guinea pigs and rats, and their total amino acid sequences are disclosed in U.S. Pat. No. 5,242,902. The growth stimulating effect of defensins on epidermal cells and fibroblast cells is disclosed in U.S. Pat. No. 5,242,902, in addition to the antimicrobial activity against Gram negative and positive bacteria and fungi. Defensins are basic peptides having about 30 amino acid residues and the common feature of six cysteine residues and three disulfide bonds.
No polypeptide isolated from the hemocytes of horseshoe crab having a similar amino acid sequence to those of the defensins has been reported. Furthermore, no nucleotide sequence corresponding to the amino acid sequence of the polypeptide of the present invention has been reported.
The inventors of the present invention further investigated the S5 peptide fraction in the isolated components of small granule fractions of horseshoe crab hemocytes and determined its full length amino acid sequence. The amino acid sequence is similar to the sequence of the C-terminus of known defensins. Further, the antimicrobial activity of the S5 peptide fraction was found to have potent antimicrobial activities against Gram positive and negative bacteria and fungi.